Multiple unfolded states of glutathione transferase bbGSTP1-1 by guanidinium chloride

Inactivation, dissociation, and unfolding of the homodimeric glutathione tr ansferase (bbGSTP1-1) from Bufo bufo embryos were investigated at equilibri um, using guanidinium chloride (GdmCI) as denaturant. Protein transitions w ere monitored by enzyme activity, intrinsic fluorescence, far UV circular d ichroism, glutaraldehyde cross-linking, and gel-filtration chromatography. At low denaturant concentrations (less than 0.5 M), reversible inactivation of the enzyme occurs. At denaturant concentrations between 0.5 and 1.5 M t he enzyme progressively dissociates into structured monomers. At higher den aturant concentrations the monomers unfold completely. Refolding studies in dicate that a total reactivation occurs only by starting from the enzyme de natured at concentrations below 0.5 M. The enzyme denatured at GdmCI concen trations higher than 0.5 NI only partially refolds. Globally our results in dicate that unfolding of the amphibian bbGSTP1-1 is a multistep process, i. e., inactivation of the structured dimer, dissociation into partially struc tured monomers, followed by complete unfolding. (C) 1999 Academic Press.